Conventional Microbiological Tests Research Articles

The clinical application of metagenomic next-generation sequencing in immunocompromised patients with severe respiratory infections in the ICU.

Early targeted antibiotic therapy is crucial for improving the prognosis of immunocompromised patients with severe respiratory infections (SRIs) in the intensive care unit (ICU). Metagenomic next-generation sequencing (mNGS) has shown significant value in pathogen detection, but research on lower respiratory tract microorganisms remains limited. This study enrolled 234 patients with SRIs in the ICU, and individuals were categorized into immunocompromised and immunocompetent groups. We compared the diagnostic performance of mNGS using bronchoalveolar lavage fluid (BALF) with conventional microbiological tests (CMTs) and analyzed the value of mNGS in immunocompromised patients with SRIs in the ICU. Among all patients, the pathogenic microorganism detection rate of mNGS was higher than that of CMTs (94.02% vs 66.67%, P < 0.05), both in the immunocompromised group (95.0% vs 58.75%, P < 0.05) and the immunocompetent group (93.51% vs 71.43%, P < 0.05). mNGS detected more pathogens than CMTs did (167 vs 51), identifying 116 organisms that were missed by CMTs. The proportion of antibiotic regimen adjustments based on mNGS results was significantly higher compared to CMTs in both the immunocompromised (70.00% vs 17.50%, P < 0.05) and immunocompetent groups (48.70% vs 15.58%, P < 0.05). In the immunocompromised group, patients who had their antibiotic treatment adjusted on mNGS results had improved prognosis, with significantly lower ICU mortality (8.93% vs 50%, P < 0.05) and 28-day mortality rates (30.36% vs 68.75%, P < 0.05) than CMTs. In the immunocompetent group, no statistically significant differences were observed in ICU mortality or 28-day mortality (20.00% vs 33.33%, P > 0.05; 42.67% vs 45.83%, P > 0.05). mNGS shows significant value in detecting pathogens in immunocompromised patients with SRIs in ICU. For immunocompromised patients who respond poorly to empirical treatment, mNGS can provide an etiological basis, helping adjust antibiotic regimens more precisely and thereby improving patient prognosis.

Comparing the diagnostic value of targeted with metagenomic next-generation sequencing in immunocompromised patients with lower respiratory tract infection

BackgroundAccurate identification of the etiology of lower respiratory tract infections (LRTI) is crucial, particularly for immunocompromised patients with more complex etiologies. The advent of next-generation sequencing (NGS) has enhanced the effectiveness of pathogen detection. However, assessments of the clinical diagnostic value of targeted NGS (tNGS) in immunocompromised patients with LRTI are limited.MethodsTo evaluate the diagnostic value of tNGS in immunocompromised patients with LRTI, a total of 88 patients, of whom 54 were immunocompromised, were enrolled. These patients underwent tNGS testing of bronchoalveolar lavage fluid (BALF). Results from both metagenomic next-generation sequencing (mNGS) and conventional microbiological tests (CMT) were also available for all participants. The performance of tNGS was assessed by comparing its findings against mNGS, CMT, and the clinical composite diagnosis.ResultsIn the cohort of 88 patients, tNGS showed comparable diagnostic value to mNGS and was significantly superior to CMT. Compared to CMT and composite reference standard, tNGS showed sensitivity of 94.55% and 90.48%, respectively. In immunocompromised patients, despite a more diverse pathogen variety, tNGS maintained similar sensitivity to mNGS and outperformed CMT. tNGS positively influenced etiologic diagnosis and antibiotic decision-making in 72.72% of cases, leading to a change in antibiotic regimen in 17.05% of cases. We also compared the detection of microbial nucleic acids by tNGS with mNGS and found that tNGS could identify 87.99% of the microbial nucleic acids identified by mNGS.ConclusionIn summary, our study demonstrated that tNGS offers promising clinical diagnostic accuracy in immunocompromised patients, as evidenced by its favorable comparison with CMT, the composite reference standard, and mNGS.

The foundation for the microbiology laboratory's essential role in diagnostic stewardship: an ASM Laboratory Practices Subcommittee report.

Diagnostic stewardship (DxS) has gained traction in recent years as a cross-disciplinary method to improve the quality of patient care while appropriately managing resources within the healthcare system. Clinical microbiology laboratorians have been highly engaged in DxS efforts to guide best practices with conventional microbiology tests and more recently with molecular infectious disease diagnostics. Laboratories can experience resistance to their role in DxS, especially when the clinical benefits, motivations for interventions, and underlying regulatory requirements are not clearly conveyed to stakeholders. Clinical laboratories must not only ensure ethical practices but also meet obligatory requirements to steward tests responsibly. In this review, we aim to support clinical microbiology laboratorians by providing the background and resources that demonstrate the laboratory's essential role in DxS. The heart of this review is to collate regulatory and accreditation requirements that, in essence, mandate DxS practices as a long-standing, core element of high-quality laboratory testing to deliver the best possible patient care. While examples of the clinical impact of DxS are plentiful in the literature, here, we focus on the operational and regulatory justification for the laboratory's role in stewardship activities.

Advantages of metagenomic next-generation sequencing in the management of ANCA-associated vasculitis patients with suspected pulmonary infection as a rule-out tool

ObjectivePulmonary infection is one of the leading causes of death in patients with ANCA-associated vasculitis (AAV). It is sometimes difficult to differentiate pulmonary infection from pulmonary involvement of vasculitis in AAV patients. Fiberoptic bronchoscopy and bronchoalveolar lavage fluid (BALF) assays are useful diagnostic methods. In addition to conventional microbiological tests (CMTs), metagenomic next-generation sequencing (mNGS) facilitates rapid and sensitive detection of various pathogens. The current study aimed to evaluate the advantages of additional BALF mNGS in the management of pulmonary infection in AAV patients.Methods27 patients with active AAV and suspected pulmonary infection whose BALF samples were tested by mNGS and CMTs and 17 active AAV patients whose BALF were tested by CMTs alone were retrospectively recruited. The results of microbiological tests, and adjustments of treatment following BALF mNGS, were described. The durations of antimicrobial treatment and in-hospital mortality in patients were compared.ResultsAmong the 27 patients whose BALF samples were tested by mNGS, 25.9% of patients did not have evidence of pathogenic microorganism in their BALF samples, 55.6% had polymicrobial infections, including bacteria, fungi and viruses. Of these 27 patients, 40.7% did not have evidence of pathogenic microorganism in their BALF or serum samples according to CMTs. Patients in the BALF mNGS/CMT group received a significantly shorter duration of antibacterial and total antimicrobial treatment than patients in the CMT alone group (17.3 ± 14.7 vs. 27.9 ± 19.0 days, P = 0.044; 18.9 ± 15.0 vs. 29.5 ± 17.7 days, P = 0.040, respectively). Fewer patients in the BALF mNGS/CMT group died than in the CMT alone group (4/27 vs. 7/17, P = 0.049).ConclusionCompared with CMT alone, additional mNGS tests may shorten the duration of antimicrobial treatment and possibly decrease death from severe infection by providing precise and quick diagnosis of infection.

Pathogen detection by targeted next-generation sequencing test in adult hematological malignancies patients with suspected infections.

Infections in patients with hematological malignancies (HM) are a significant cause of morbidity and mortality. Timely and effective empirical anti-infective treatment can reduce the infection-related mortality rate. Targeted next-generation sequencing (tNGS) offers a rapid diagnostic approach for identifying diverse pathogens in these patients. However, relevant research is still limited to adult patients with HM. We conducted a retrospective analysis of adult HM patients admitted to our hospital from March 2023 to September 2023, focusing on their clinical characteristics and the results of both tNGS and conventional microbiological tests (CMTs). We evaluated the performance of tNGS and CMTs in pathogenic diagnosis and described the distribution characteristics of pathogens in adult HM patients with infections. The study included 209 samples collected from 137 patients. Results showed that the overall pathogen detection rate differed significantly between tNGS and CMTs (60.3% vs. 24.4%, p < 0.001). The sensitivity (69.7% vs. 35.9%), negative predictive value (NPV) (48.2% vs. 42.4%), and accuracy (66.5% vs. 56.5%) of pathogen detection were notably superior with tNGS compared to CMTs. Among the 142 samples with clinically diagnosed infections, tNGS combined with CMTs identified a definite or probable microbial etiology in 114 samples (80.3%). Of the 36 samples with concordant positive results from both tNGS and CMTs, 72.2% (26/36) exhibited full or partial agreement. Our study further showed the highest detection rate for viral infections (57.0%), predominantly for Epstein-Barr virus (DNA-V, 18.3%). Followed by bacterial infections (46.5%), the detection rate of Gram-negative bacteria (G+, 35.9%) was higher than that of Gram-positive bacteria (G-, 21.8%) in this study. Klebsiella pneumoniae (G-, 12.7%) had the highest detection rate among these emerging bacteria, followed by Pseudomonas aeruginosa (G-, 10.6%) and Enterococcus faecium (G+, 7.7%). Bacterial-viral coinfections were the most common type of mixed infection (35.5%). In conclusion, tNGS outperforms CMTs in both sensitivity and pathogen spectrum. Therefore, it can serve as an adjunct to CMTs to facilitate the precise adjustment of anti-infective regimens for adult HM patients. Our findings establish a basis for formulating empirical anti-infective therapy strategies tailored to the pathogen distribution in this patient population.

Clinical evaluation of a highly multiplexed CRISPR-based diagnostic assay for diagnosing lower respiratory tract infection: a prospective cohort study

Objective Accurate and rapid identification of causative pathogens is essential to guide the clinical management of lower respiratory tract infections (LRTIs). Here we conducted a single-centre prospective study in 284 patients suspected of lower respiratory tract infections to evaluate the utility of a nucleic acid test based on highly multiplexed polymerase chain reaction (PCR) and CRISPR-Cas12a. Methods We determined the analytical and diagnostic performance of the CRISPR assay using a combination of reference standards, including conventional microbiological tests (CMTs), metagenomic Next-Generation Sequencing (mNGS), and clinical adjudication by a panel of experts on infectious diseases and microbiology. Results The CRISPR assay showed a higher detection rate (63.0%) than conventional microbiological tests (38.4%) and was lower than metagenomic Next-Generation Sequencing (72.9%). In detecting polymicrobial infections, the positivity rate of the CRISPR assay (19.4%) was higher than conventional microbiological tests (3.5%) and lower than metagenomic Next-Generation Sequencing (28.9%). The overall diagnostic sensitivity of the CRISPR assay (67.8%) was higher than conventional microbiological tests (41.8%), and lower than metagenomic Next-Generation Sequencing (93.2%). Conclusions Considering the low cost, ease of operation, short turnaround time, and broad range of pathogens detected in a single test, the CRISPR assay has the potential to be implemented as a screening tool for the aetiological diagnosis of lower respiratory tract infections patients, especially in cases where atypical bacteria or coinfections are suspected.

Enhancing lower respiratory tract infection diagnosis: implementation and clinical assessment of multiplex PCR-based and hybrid capture-based targeted next-generation sequencing

Shotgun metagenomic next-generation sequencing (mNGS) is widely used to detect pathogens in bronchoalveolar lavage fluid (BALF). However, mNGS is complex and expensive. This study explored the feasibility of targeted next-generation sequencing (tNGS) in distinguishing lower respiratory tract infections in clinical practice. We used 229 retrospective BALF samples to establish thresholds and diagnostic values in a prospective cohort of 251 patients. After target pathogen selection, primer and probe design, optimization experiments, and bioinformatics analysis, multiplex PCR-based tNGS (mp-tNGS) and hybrid capture-based tNGS (hc-tNGS), targeting 198 and 3060 pathogens (DNA and RNA co-detection workflow) were established and performed. mp-tNGS and hc-tNGS took 10.3 and 16h, respectively, with low sequencing data sizes of 0.1M and 1M reads, and test costs reduced to a quarter and half of mNGS. The LoDs of mp-tNGS and hc-tNGS were 50-450CFU/mL. mp-tNGS and hc-tNGS were highly accurate, with 86.5% and 87.3% (vs. 85.5% for mNGS) sensitivities and 90.0% and 88.0% (vs. 92.1% for mNGS) specificities. tNGS detection rates for casual pathogens were 84.3% and 89.5% (vs. 88.5% for mNGS), significantly higher than conventional microbiological tests (P<0.001). In seven samples, tNGS detected Pneumocystis jirovecii, a fungus not detected by mNGS. Whereas mNGS detected six samples with filamentous fungi (Rhizopus oryzae, Aureobasidium pullulans, Aspergillus niger complex, etc.) which missed by tNGS. The anaerobic bacteria as pathogen in eight samples was failed to detect by mp-tNGS. tNGS may offer a new, broad-spectrum, rapid, accurate and cost-effective approach to diagnosing respiratory infections. National Natural Science Foundation of China (81625014 and 82202535).

Molecular Evaluation of Aminoglycoside Resistance and Biofilm Formation Potential in Escherichia coli Isolates Collected from Hospitalized Patients

Background: Resistance to antibiotics and the ability to develop biofilms, two main virulence determinants of Escherichia coli, play a crucial role in the persistence of infections. Objectives: The aim of this study was to evaluate aminoglycoside resistance and biofilm formation potential in E. coli isolates collected from hospitalized patients in the Southwest of Iran. Methods: A total of 70 E. coli clinical isolates from different specimens were collected from Ahvaz teaching hospitals affiliated with Ahvaz Jundishapur University of Medical Sciences. All the isolates were identified as E. coli using conventional microbiological tests. Susceptibility to antibiotics was determined using the Kirby–Bauer disk diffusion method. Biofilm formation was assessed using the microtiter plate method. Finally, PCR was conducted to detect virulence gene determinants, including fimbrial genes, aminoglycoside modifying enzymes (AMEs), and 16S rRNA methylase (RMTase) genes. Results: Among aminoglycoside antibiotics, E. coli isolates showed the highest and lowest resistance rates to tobramycin (TOB; 51.4%) and gentamicin (GEN; 24.2%), respectively. Simultaneous resistance to GEN, amikacin, and TOB was observed in 28.5% of the isolates, representing the most common antibiotic resistance pattern. The prevalence of strong biofilm producers was higher in the extensively drug-resistant (XDR) phenotype group compared to the multiple drug-resistant (MDR) group (76.1% vs. 23.8%). Among the 36 isolates resistant to at least one of the aminoglycoside antibiotics, 36.1% had AME-related genes, either alone or in various combinations. Most isolates harboring AME genes were also positive for the presence of biofilm-related genes, including ecpA and fimA. Conclusions: The most frequent AME-related genes were ant(2”)-Ia and aph(3’)-Ia, followed by aac(3’)-IIa. The findings of the present study provide probable evidence that GEN is an effective aminoglycoside against biofilm-producing and antibiotic-resistant E. coli isolates.

Multiplex real-time PCR in non-invasive respiratory samples to reduce antibiotic use in community-acquired pneumonia: a randomised trial

We assessed whether multiplex real-time PCR plus conventional microbiological testing is safe and more effective than conventional microbiological testing alone for reducing antibiotic use in community-acquired pneumonia (CAP). In this randomised trial, we recruited adults hospitalised with CAP at four Spanish hospitals. Patients were randomly assigned (1:1) to undergo either multiplex real-time PCR in non-invasive respiratory samples plus conventional microbiological testing or conventional microbiological testing alone. The primary endpoint was antibiotic use measured by days of antibiotic therapy (DOT). Between February 20, 2020, and April 24, 2023, 242 patients were enrolled; 119 were randomly assigned to multiplex real-time PCR plus conventional microbiological testing and 123 to conventional microbiological testing alone. All but one of the patients allocated to multiplex real-time PCR plus conventional microbiological testing underwent PCR, which was performed in sputum samples in 77 patients (65.2%) and in nasopharyngeal swabs in 41 (34.7%). The median DOT was 10.04 (IQR 7.98, 12.94) in the multiplex PCR plus conventional microbiological testing group and 11.33 (IQR 8.15, 16.16) in the conventional microbiological testing alone group (difference −1.04; 95% CI, −2.42 to 0.17; p = 0.093). No differences were observed in adverse events and 30-day mortality. Our findings do not support the routine implementation of multiplex real-time PCR in the initial microbiological testing in hospitalised patients with CAP. Clinicaltrials.gov registration: NCT04158492.

Metagenomic Next-Generation Sequencing in the Diagnosis of Pulmonary Infections after Allogeneic Hematopoietic Stem Cell Transplantation

Early and accurate identification of pathogens in pulmonary infections after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is critically important. The clinical usefulness of metagenomic next-generation sequencing (mNGS) in the diagnosis of pulmonary infections after allo-HSCT remains under discussion. This multicenter retrospective study was conducted to compare mNGS and conventional microbiological tests (CMTs) in identifying the pathogens of pulmonary infections in allo-HSCT recipients. One hundred forty allo-HSCT recipients with suspected pulmonary infections who underwent bronchoscopy were included. mNGS and CMTs performed on bronchoalveolar lavage fluid specimens showed 71.4% positivity on mNGS compared to 55.0% positivity on CMTs. mNGS identified 182 pathogens, including bacteria (n = 88), fungi (n = 35) and viruses (n = 59), compared to 106 pathogens detected by CMTs (bacteria, n = 31; fungi, n = 24; viruses, n = 51). Pulmonary infection was finally diagnosed in 98 patients, including 22 bacterial, 7 fungal, 18 viral, and 48 mixed infections and 3 infections with an unknown pathogen. Mixed infections were identified in 50.5% of the patients with pulmonary infection. The sensitivity of mNGS and CMTs for diagnosing pulmonary infections was 88.8% and 69.4%, respectively (P = .001), and the specificity were 81.0% and 85.7%, respectively (P = .688). Our findings suggest that mNGS may be a promising technology for diagnosing pulmonary infections in allo-HSCT recipients.

Microbiological characteristics of the lower airway in adults with bronchiectasis: a prospective cohort study

BackgroundMicrobial infection and colonization are frequently associated with disease progression and poor clinical outcomes in bronchiectasis. Identification of pathogen spectrum is crucial for precision treatment at exacerbation of bronchiectasis.MethodsWe conducted a prospective cohort study in patients with bronchiectasis exacerbation onset and stable state. Bronchoalveolar lavage fluid (BALF) was collected for conventional microbiological tests (CMTs) and metagenomic Next-Generation Sequencing (mNGS). Bronchiectasis patients were monitored for documenting the time to the next exacerbation during longitudinal follow-up.ResultsWe recruited 168 eligible participants in the exacerbation cohorts, and 38 bronchiectasis patients at stable state at longitudinal follow-up. 141 bronchiectasis patients at exacerbation onset had definite or probable pathogens via combining CMTs with mNGS reports. We identified that Pseudomonas aeruginosa, non-tuberculous mycobacteria, Haemophilus influenzae, Nocardia spp, and Staphylococcus aureus were the top 5 pathogens with a higher detection rate in our cohorts via combination of CMTs and mNGS analysis. We also observed strong correlations of Pseudomonas aeruginosa, Haemophilus influenzae, non-tuberculous mycobacteria with disease severity, including the disease duration, Bronchiectasis Severity Index, and lung function. Moreover, the adjusted pathogenic index of potential pathogenic microorganism negatively correlated (r = -0.7280, p < 0.001) with the time to the next exacerbation in bronchiectasis.ConclusionWe have revealed the pathogenic microbial spectrum in lower airways and the negative correlation of PPM colonization with the time to the next exacerbation in bronchiectasis. These results suggested that pathogens contribute to the progression of bronchiectasis.

Effect of Metagenomic Next-Generation Sequencing on Clinical Outcomes of Patients With Severe Community-Acquired Pneumonia in the ICU: A Multicenter, Randomized Controlled Trial

Metagenomic next-generation sequencing (mNGS) was previously established as a method that can increase the pathogen identification rate in patients with severe community-acquired pneumonia (SCAP). What is the impact on clinical outcomes of mNGS of BAL fluid (BALF) in patients with SCAP in the ICU? A multicenter, randomized controlled, open-label clinical trial was conducted in 10 ICUs. Patients were randomized in a 1:1 ratio to undergo BALF assessment with conventional microbiological tests (CMTs) only (ie, the CMT group) or BALF assessment with both mNGS and CMTs (ie, the mNGS group). The primary outcome was the time to clinical improvement, defined as the time from randomization to either an improvement of two points on a six-category ordinal scale or discharge from the ICU, whichever occurred first. A total of 349 patients were randomized to treatment between January 1, 2021, and November 18, 2022; 170 were assigned to the CMT group and 179 to the mNGS group. In the intention-to-treat analysis, the time to clinical improvement was better in the mNGS group than in the CMT group (10days vs13days; difference, -2.0days; 95%CI, -3.0 to 0.0days). Similar results were obtained in the per-protocol analysis. The proportion of patients with clinical improvement within 14days was significantly higher in the mNGS group (62.0%) than in the CMT group (46.5%). There was no significant difference in other secondary outcomes. Compared with the use of CMTs alone, mNGS combined with CMTs reduced the time to clinical improvement for patients with SCAP. ChiCTR2000037894.

The diagnostic value of bronchoalveolar lavage fluid metagenomic next-generation sequencing in critically ill patients with respiratory tract infections.

Metagenomic next-generation sequencing (mNGS) is an unbiased and rapid method for detecting pathogens. This study enrolled 145 suspected severe pneumonia patients who were admitted to the Affiliated Hospital of Jining Medical University. This study primarily aimed to determine the diagnostic performance of mNGS and conventional microbiological tests (CMTs) using bronchoalveolar lavage fluid samples for detecting pathogens. Our findings indicated that mNGS performed significantly higher sensitivity (97.54% vs 28.68%, P < 0.001), coincidence (90.34% vs 35.17%, P < 0.001), and negative predictive value (80.00% vs 13.21%, P < 0.001) but performed lower specificity than CMTs (52.17% vs 87.5%, P < 0.001). Streptococcus pneumoniae as the most common bacterial pathogen had the largest proportion (22.90%, 30/131) in this study. In addition to bacteria, fungi, and virus, mNGS can detect a variety of atypical pathogens such as Mycobacterium tuberculosis and non-tuberculous. Mixed infections were common in patients with severe pneumonia, and bacterial-fungal-viral-atypical pathogens were the most complicated infection. After adjustments of antibiotics based on mNGS and CMTs, the clinical manifestation improved in 139 (95.86%, 139/145) patients. Our data demonstrated that mNGS had significant advantage in diagnosing respiratory tract infections, especially atypical pathogens and fungal infections. Pathogens were detected timely and comprehensively, contributing to the adjustments of antibiotic treatments timely and accurately, improving patient prognosis and decreasing mortality potentially.IMPORTANCEMetagenomic next-generation sequencing using bronchoalveolar lavage fluid can provide more comprehensive and accurate pathogens for respiratory tract infections, especially when considering the previous usage of empirical antibiotics before admission or complicated clinical presentation. This technology is expected to play an important role in the precise application of antimicrobial drugs in the future.

A single-center, retrospective study of hospitalized patients with lower respiratory tract infections: clinical assessment of metagenomic next-generation sequencing and identification of risk factors in patients

IntroductionLower respiratory tract infections(LRTIs) in adults are complicated by diverse pathogens that challenge traditional detection methods, which are often slow and insensitive. Metagenomic next-generation sequencing (mNGS) offers a comprehensive, high-throughput, and unbiased approach to pathogen identification. This retrospective study evaluates the diagnostic efficacy of mNGS compared to conventional microbiological testing (CMT) in LRTIs, aiming to enhance detection accuracy and enable early clinical prediction.MethodsIn our retrospective single-center analysis, 451 patients with suspected LRTIs underwent mNGS testing from July 2020 to July 2023. We assessed the pathogen spectrum and compared the diagnostic efficacy of mNGS to CMT, with clinical comprehensive diagnosis serving as the reference standard. The study analyzed mNGS performance in lung tissue biopsies and bronchoalveolar lavage fluid (BALF) from cases suspected of lung infection. Patients were stratified into two groups based on clinical outcomes (improvement or mortality), and we compared clinical data and conventional laboratory indices between groups. A predictive model and nomogram for the prognosis of LRTIs were constructed using univariate followed by multivariate logistic regression, with model predictive accuracy evaluated by the area under the ROC curve (AUC).Results(1) Comparative Analysis of mNGS versus CMT: In a comprehensive analysis of 510 specimens, where 59 cases were concurrently collected from lung tissue biopsies and BALF, the study highlights the diagnostic superiority of mNGS over CMT. Specifically, mNGS demonstrated significantly higher sensitivity and specificity in BALF samples (82.86% vs. 44.42% and 52.00% vs. 21.05%, respectively, p < 0.001) alongside greater positive and negative predictive values (96.71% vs. 79.55% and 15.12% vs. 5.19%, respectively, p < 0.01). Additionally, when comparing simultaneous testing of lung tissue biopsies and BALF, mNGS showed enhanced sensitivity in BALF (84.21% vs. 57.41%), whereas lung tissues offered higher specificity (80.00% vs. 50.00%). (2) Analysis of Infectious Species in Patients from This Study: The study also notes a concerning incidence of lung abscesses and identifies Epstein-Barr virus (EBV), Fusobacterium nucleatum, Mycoplasma pneumoniae, Chlamydia psittaci, and Haemophilus influenzae as the most common pathogens, with Klebsiella pneumoniae emerging as the predominant bacterial culprit. Among herpes viruses, EBV and herpes virus 7 (HHV-7) were most frequently detected, with HHV-7 more prevalent in immunocompromised individuals. (3) Risk Factors for Adverse Prognosis and a Mortality Risk Prediction Model in Patients with LRTIs: We identified key risk factors for poor prognosis in lower respiratory tract infection patients, with significant findings including delayed time to mNGS testing, low lymphocyte percentage, presence of chronic lung disease, multiple comorbidities, false-negative CMT results, and positive herpesvirus affecting patient outcomes. We also developed a nomogram model with good consistency and high accuracy (AUC of 0.825) for predicting mortality risk in these patients, offering a valuable clinical tool for assessing prognosis.ConclusionThe study underscores mNGS as a superior tool for lower respiratory tract infection diagnosis, exhibiting higher sensitivity and specificity than traditional methods.

Emerging Trends of Immunosensors Development for Detection of Food Toxins

The present study highlights the ongoing threat of foodborne illnesses to public health, primarily caused by bacterial pathogens. Despite advancements in conventional microbiological testing techniques, which are sensitive but time-consuming, challenges remain in ensuring timely detection of contaminants throughout the food supply chain. The Hazard Analysis Critical Control Point (HACCP) system is recognized as a more effective approach to ensuring food safety, emphasizing proactive identification and control of hazards at critical points in production. Emerging technologies like quantitative polymerase chain reaction (PCR) and biosensors offer faster and more accurate detection methods, although with certain limitations. Biosensors such as ELISA, SPR, and electrochemical immunosensors, in particular, show promise due to their high sensitivity and specificity, enabling rapid detection of a wide range of contaminants. This paper underscores the importance of integrating advanced technologies with established food safety protocols to enhance the safety and quality of food products, benefiting consumers, producers, and regulatory agencies alike.

The impact of neutrophil count on the results of metagenomic next-generation sequencing in immunocompromised febrile children

Metagenomic next-generation sequencing (mNGS) has revolutionized the detection of pathogens, particularly in immunocompromised individuals such as pediatric patients undergoing intensive chemotherapy and hematopoietic stem cell transplantation. This study aims to explore the impact of neutrophil count on the diagnostic efficacy of mNGS in diagnosing infections in pediatric patients with febrile diseases. We conducted a retrospective analysis of pediatric patients with febrile diseases in the hematology/oncology department from January 2019 to September 2022. The study included 387 patients with 516 febrile episodes. Analyzing data from 516 pediatric cases, our study found that 70.7 % had febrile neutropenia (FN) and 29.3 % had febrile without neutropenia (FWN). mNGS demonstrated a high positive detection rate of 84.9 %, compared to 29.7 % for conventional microbiological tests (CMT). While the positive detection rates of mNGS were similar in both FN and FWN groups, bacterial pathogens were more frequently detected in FN patients. Furthermore, the rate of identifying a “probable” microbial etiology was lower in the FN group (46.8 %) compared to the FWN group (65.6 %, p＜0.001). When analyzing the types of organisms and specimens, the “probable” identification rates were particularly lower for viruses and fungi detected by mNGS, as well as in blood and nasopharyngeal swab samples. These findings underscore the significant influence of neutrophil counts on mNGS results in pediatric febrile patients and highlight the necessity for tailored diagnostic approaches in this population.

Microbiological Profile of Patients with Aspiration Pneumonia Identified by Combined Detection Methods.

Aspiration pneumonia (AP) challenges public health globally. The primary aim of this study was to ascertain the microbiological profile characteristics of patients with AP evaluated by combined detection methods, including conventional microbiological tests (CMTs), chips for complicated infection detection (CCID), and metagenomic next-generation sequencing (mNGS). From June 2021 to March 2022, a total of thirty-nine patients with AP or community-acquired pneumonia with aspiration risk factors (AspRF-CAP) from 3 hospitals were included. Respiratory specimens, including bronchoalveolar lavage fluid (BALF), sputum, and tracheal aspirate, were collected for microorganism detection. Patients with AP were more inclined to be older, to have a shorter duration from illness onset to admission, to have a higher prevalence of different underlying diseases, particularly diabetes mellitus, chronic heart disease, and cerebrovascular disease, and to have a higher CURB-65 score (all P < 0.05). A total of 213 and 31 strains of microorganisms were detected in patients with AP and AspRF-CAP, respectively. The most common pathogens in AP were Corynebacterium striatum (17/213, 7.98%), Pseudomonas aeruginosa (15/213, 7.04%), Klebsiella pneumoniae (15/213, 7.04%), and Candida albicans (14/213, 6.57%). Besides, the most common pathogens in AspRF-CAP were Candida albicans (5/31, 16.13%), Pseudomonas aeruginosa (3/31, 9.68%) and Klebsiella pneumoniae (3/31, 9.68%). Moreover, Klebsiella pneumoniae (7/67, 10.45%) and Candida glabrata (5/67, 7.46%) were the most common pathogens among the 9 non-survived patients with AP. The prevalent pathogens detected in cases of AP were Corynebacterium striatum, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Candida albicans. Early combined detection methods for patients with AP enhance the positive detection rate of pathogens and potentially expedites the initiation of appropriate antibiotic therapeutic strategies.

Clinical application of nanopore-targeted sequencing technology in bronchoalveolar lavage fluid from patients with pulmonary infections.

The rapid and effective identification of pathogens in patients with pulmonary infections has posed a persistent challenge in medicine, with conventional microbiological tests (CMTs) proving time-consuming and less sensitive, hindering early diagnosis of respiratory infections. While there has been some research on the clinical performance of targeted sequencing technologies, limited focus has been directed toward bronchoalveolar lavage fluid (BALF). This study primarily evaluates the pathogen detection capabilities of nanopore-targeted sequencing (NTS) in BALF, providing a comprehensive analysis. The retrospective study, spanning from January 2022 to November 2023, includes 223 patients exclusively sourced from a single center. We conducted a detailed comparative analysis among NTS, targeted next-generation sequencing (tNGS), and CMTs. Initially, we compared the detection capabilities of NTS and tNGS and found no significant differences in their sensitivity and specificity. Specifically, we observed that the sensitivity of NTS was significantly higher than that of CMTs (74.83% vs 33.11%, P < 0.001). Furthermore, NTS exhibited a higher positivity rate in common pulmonary infections (62.88% vs. 23.48%) and in clinically suspected tuberculosis patients compared to CMTs (87.18% vs. 48.72%). Additionally, NTS showed less susceptibility to antibiotic interference, indicating a more sensitive detection capability, especially in detecting fastidious organisms. It complements GeneXpert in tuberculosis diagnosis and offers excellent advantages in identifying pathogens challenging for CMTs, such as non-tuberculous mycobacteria and viruses. Moreover, NTS significantly shortens the reporting time and is only a quarter of the cost of metagenomic next-generation sequencing. Clearly, NTS can facilitate faster and more cost-effective early diagnosis of respiratory infections.IMPORTANCEThis study holds paramount significance in advancing the field of respiratory infection diagnostics. By assessing the pathogen detection capabilities in bronchoalveolar lavage fluid (BALF) of patients with pulmonary infections, we illuminate the promising potential of nanopore-targeted sequencing (NTS). The findings underscore NTS as a comparable yet distinct alternative to traditional methods like comprehensive conventional microbiological tests (CMTs). Notably, NTS demonstrates a pivotal edge, expanding the spectrum of identified pathogens, particularly excelling in the detection of challenging entities like non-tuberculous mycobacteria and viruses. The study also highlights the complementary role of NTS alongside GeneXpert in the identification of tuberculosis, providing a comprehensive overview of the diagnostic landscape for respiratory infections. This insight carries significant implications for clinicians seeking rapid, cost-effective, and accurate diagnostic tools in the realm of pulmonary infections.

Exploring the pathogen diagnosis and prognostic factors of severe COVID-19 using metagenomic next-generation sequencing: A retrospective study.

This study aimed to identify pathogens and factors that predict the outcome of severe COVID-19 by utilizing metagenomic next-generation sequencing (mNGS) technology. We retrospectively analyzed data from 56 severe COVID-19 patients admitted to our hospital between December 2022 and March 2023. We analyzed the pathogen types and strains detected through mNGS and conventional microbiological testing and collected general patient information. In this study, 42 pathogens were detected using mNGS and conventional microbiological testing. mNGS had a significantly higher detection rate of 90.48% compared to 71.43% for conventional testing (P=0.026). A total of 196 strains were detected using both methods, with a significantly higher detection rate of 70.92% for mNGS compared to 49.49% for conventional testing (P=0.000). The 56 patients were divided into a survival group (33 cases) and a death group (23 cases) based on clinical outcomes. The survival group had significantly lower age, number of pathogens detected by mNGS, number of pathogens detected by conventional testing, APACHE-II score, SOFA score, high-sensitivity troponin, creatine kinase-MB subtype, and lactate dehydrogenase compared to the death group (P<0.05). Multivariate logistic regression analysis showed that these factors were risk factors for mortality in severe COVID-19 patients (P<0.05). In contrast, ROC curve analysis revealed that these factors had diagnostic values for mortality, with AUC values ranging from 0.657 to 0.963. The combined diagnosis of these indicators had an AUC of 0.924. The use of mNGS technology can significantly enhance the detection of pathogens in severe cases of COVID-19 and also has a solid ability to predict clinical outcomes.

Application value of metagenomic next-generation sequencing in hematological patients with high-risk febrile neutropenia.

Metagenomic next-generation sequencing (mNGS) is a novel non-invasive and comprehensive technique for etiological diagnosis of infectious diseases. However, its practical significance has been seldom reported in the context of hematological patients with high-risk febrile neutropenia, a unique patient group characterized by neutropenia and compromised immune responses. This retrospective study evaluated the results of plasma cfDNA sequencing in 164 hematological patients with high-risk febrile neutropenia. We assessed the diagnostic efficacy and clinical impact of mNGS, comparing it with conventional microbiological tests. mNGS identified 68 different pathogens in 111 patients, whereas conventional methods detected only 17 pathogen types in 36 patients. mNGS exhibited a significantly higher positive detection rate than conventional methods (67.7% vs. 22.0%, P < 0.001). This improvement was consistent across bacterial (30.5% vs. 9.1%), fungal (19.5% vs. 4.3%), and viral (37.2% vs. 9.1%) infections (P < 0.001 for all comparisons). The anti-infective treatment strategies were adjusted for 51.2% (84/164) of the patients based on the mNGS results. mNGS of plasma cfDNA offers substantial promise for the early detection of pathogens and the timely optimization of anti-infective therapies in hematological patients with high-risk febrile neutropenia.